The mammalian lung´s structural design is optimized to serve its main function: gas exchange. It takes place in the alveolar region (parenchyma) where air and blood are brought in close proximity over a large surface. Air reaches the alveolar lumen via a conducting airway tree. Blood flows in a capillary network embedded in inter-alveolar septa. The barrier between air and blood consists of a continuous alveolar epithelium (a mosaic of type I and type II alveolar epithelial cells), a continuous capillary endothelium and the connective tissue layer in-between. By virtue of its respiratory movements, the lung has to withstand mechanical challenges throughout life. Alveoli must be protected from over-distension as well as from collapse by inherent stabilizing factors. The mechanical stability of the parenchyma is ensured by two components: a connective tissue fiber network and the surfactant system. The connective tissue fibers form a continuous tensegrity (tension + integrity) backbone consisting of axial, peripheral and septal fibers. Surfactant (surface active agent) is the secretory product of type II alveolar epithelial cells and covers the alveolar epithelium as a biophysically active thin and continuous film. Here, we briefly review the structural components relevant for gas exchange. Then we describe our current understanding of how these components function under normal conditions and how lung injury results in dysfunction of alveolar micromechanics finally leading to lung fibrosis.

The growing awareness of the importance of accurate morphometry in lung research has recently motivated the publication of guidelines set forth by a combined task force of the American Thoracic Society and the European Respiratory Society ( 20 ). This official ATS/ERS Research Policy Statement provides general recommendations on which stereological methods are to be used in quantitative microscopy of the lung. However, to integrate stereology into a particular experimental study design, investigators are left with the problem of how to implement this in practice. Specifically, different animal models of human lung disease require the use of different stereological techniques and may determine the mode of lung fixation, tissue processing, preparation of sections, and other things. Therefore, the present companion articles were designed to allow a short practically oriented introduction into the concepts of design-based stereology (Part 1) and to provide recommendations for choosing the most appropriate methods to investigate a number of important disease models (Part 2). Worked examples with illustrative images will facilitate the practical performance of equivalent analyses. Study algorithms provide comprehensive surveys to ensure that no essential step gets lost during the multistage workflow. Thus, with this review, we hope to close the gap between theory and practice and enhance the use of stereological techniques in pulmonary research.

Preservation of original tissue dimensions is an essential prerequisite for morphometric studies. Shrinkage occurring during tissue processing for histology may severely influence the appearance of structures seen under the microscope and stereological calculations. Therefore, shrinkage has to be avoided so that estimates obtained by application of unbiased stereology are indeed unbiased. The present study investigates the alterations of tissue dimensions of mouse lung samples during processing for histology. Different fixatives as well as embedding protocols are considered. Mouse lungs were fixed by instillation of either 4% formalin or a mixture of 1.5% glutaraldehyde/1.5% formaldehyde. Tissue blocks were sampled according to principles of stereology for embedding in paraffin, glycol methacrylate without treatment with osmium tetroxide and uranyl acetate, and glycol methacrylate including treatment with osmium tetroxide and uranyl acetate before dehydration. Shrinkage was investigated by stereological measurements of dimensional changes of tissue cut faces. Results show a shrinkage of the cut face areas of roughly 40% per lung during paraffin embedding, 30% during “simple” glycol methacrylate embedding, and <3% during osmium tetroxide/uranyl acetate/glycol methacrylate embedding. Furthermore, the superiority of the glutaraldehyde-containing fixative regarding shrinkage is demonstrated. In conclusion, the use of a glutaraldehyde-containing fixative and embedding in glycol methacrylate with previous treatment of the samples with osmium tetroxide and uranyl acetate before dehydration is recommended for stereological studies of the mouse lung.

The surfactant system of the lung has essential biophysical and immunomodulatory functions. Only at the electron microscopic level does surfactant reveal its morphological complexity--and beauty. Therefore, morphological tools are indispensible to characterize the surfactant system in health and disease. Stereology provides the gold standard for obtaining quantitative (morphometric) data in microscopy. The combination of microscopy and stereology allows for qualitative and quantitative analysis of the intraalveolar as well as the intracellular surfactant pool, both in its preserved microorganization and localization within the lung. Surfactant-producing alveolar epithelial type II cells can be counted and sampled for size estimation with physical disectors at a high magnification light microscopic level. The number of their surfactant storing lamellar bodies can be estimated using physical disectors at the electron microscopic level. Electron tomography allows for high resolution 3D visualization of lamellar body fusion pores. Intraalveolar surfactant subtypes can be quantitated in situ, thus reflecting the functional state of the intraalveolar surfactant pool. By immunoelectron microscopy, surfactant protein distribution can be analyzed. These methods allow for a comprehensive quantitative analysis of surfactant (ultra-)structure. Here, we give an overview on the analysis of the normal and disordered surfactant system by electron microscopy and stereology.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by patchy scarring of the distal lung with limited therapeutic options and poor prognosis. Here, we show that conditional deletion of the ubiquitin ligase Nedd4-2 (Nedd4l) in lung epithelial cells in adult mice produces chronic lung disease sharing key features with IPF including progressive fibrosis and bronchiolization with increased expression of Muc5b in peripheral airways, honeycombing and characteristic alterations in the lung proteome. NEDD4-2 is implicated in the regulation of the epithelial Na⁺ channel critical for proper airway surface hydration and mucus clearance and the regulation of TGFβ signaling, which promotes fibrotic remodeling. Our data support a role of mucociliary dysfunction and aberrant epithelial pro-fibrotic response in the multifactorial disease pathogenesis. Further, treatment with the anti-fibrotic drug pirfenidone reduced pulmonary fibrosis in this model. This model may therefore aid studies of the pathogenesis and therapy of IPF.